MMPs Inhibitor Enhance Anti-CTLA-4Antibody Immunotherapy Of Breast Cancer By Reprogramming Tumor Microenvironment

Breast cancer is now the most common cancer in women. Blocking CTLA4isa promising anti-tumor immunotherapy treatment. Preclinical Researches show thatanti-CTLA-4antibody (a-CTLA-4) promotes the activation and proliferation of Tlymphocytes, breaking tumor immune tolerance, promoting tumor regression. But,a-CTLA-4has been failing to metastatic malignant tumor. In recent years, more andmore research shows that the tumor microenvironment (TME) becomes the keyfactor of tumor immune escape and regulating the tumor sensitivity to anticancerdrugs. With TME for the treatment of target studies offer a new hope for thetreatment of malignant tumor. Matrix metalloproteinases (MMPs) act on a variety ofcomponents in TME, promoting the occurrence and development of tumor.Therefore, we try to use MMPs inhibitor (MMPI) to restrain activity of MMPs inorder to improve the tumor microenvironment, improve the effect of a-CTLA4treatment.We establish breast cancer animal model by subcutaneous injection of4T1,which is a cell line with higher metastasis potential. Mouse divide into four group:vehicle, a–CTLA-4, MMPI, a–CTLA-4+MMPI. The two drugs were administrationfrom the day3to the day21. The tumor was measured by caliper and Ultrasound.After treatment, tumor metastases were measured. The function of liver and kidneyof mice was test by biochemical analyzer. Then the mice were killed and the immunecells of spleen and bone marrow were measured by flow cytometry, usingimmunofluorescence and immunohistochemistry to detect the immune cells andneovascularization in the TME. The experimental results are as follows:1. MMPs inhibitor of CTLA4antibodies effect for the treatment of breast cancer growth and metastasis in mice1.1Compared to MMPI monotherapy or vehicle, MMPs inhibitor anda-CTLA-4treatment delays tumor growth (P<0.05).1.2Mice from each group have tumor metastases of lung tissue, ranging fromnumber and size through macroscopic observation. The results show that comparedwith vehicle group, lung metastases of mice in the a-CTLA4and MMPI groupreduce significantly（P<0.05）, but there is no statistical significance betweena-CTLA4and vehicle（P>0.05）.The number of liver and lung metastases wascounted by microscope. The results show that compared with vehicle group, a-CTLA4and MMPI reduce significantly（P<0.001）,but there is no statistical significancebetween a-CTLA4and vehicle（P>0.05）.1.3function of liver and kidney of mice in each group has no statisticalsignificance compared with normal mice（P>0.05）.2. MMPs inhibitor of CTLA4antibodies in the treatment of breast cancer inmice immunological indexes2.1CD4+T, CD8+T cell and CD8+T/CD4+T: Compared to vehicle,monotherapy, Combination a-CTLA-4and MMPI increases the percentage ofCD4+T CD8+T and the ratio of CD8+T/CD4+T in spleen. There is there is astatistical significance between MMPI and vehicle (P<0.05).2.2Treg cell, Th17and MDSCs: Compared to vehicle, a-CTLA-4and MMPIinhibits the percentage of Treg cell, MDSCs, the ratio of Treg cell and Th17(P<0.05),but there is no statistical significance between a-CTLA4and vehicle（P>0.05）.Thepercentage of Th17in a-CTLA-4and MMPI reduces compared with vehicle(P<0.05).3MMPS inhibitor of CTLA4antibodies effect for the treatment of breastcancer in mice tumor microenvironment3.1CD4+T, CD8+T cell and CD8+T/CD4+T: Compared to vehicle, a-CTLA-4,Combination a-CTLA-4and MMPI increases the percentage of CD4+T CD8+T andthe ratio of CD8+T/CD4+T in tumor. There is there is a statistical significance between MMPI+a-CTLA-4group and a-CTLA-4(P<0.05).3.2Treg cell, Th17and MDSCs: Compared to vehicle, a-CTLA-4and MMPIinhibits the percentage of Treg cell, MDSCs, Th17(P<0.05), but there is no statisticalsignificance between a-CTLA4and vehicle（P>0.05）. Compare with vehicle,monotherapy, the ratio of Treg cell and Th17in mouse of a-CTLA-4and MMPI hasnot statistical significance (P>0.05).3.3Compared to vehicle, a-CTLA-4treatment dose not reduce MVD in tumor,but has statistical significance with MMPI and a-CTLA-4(P<0.01). There is there isa statistical significance between MMPI+a-CTLA-4group and a-CTLA-4(P<0.05).3.4Correlation analysis showed by Spearman that, there is a negativecorrelation between MVD and the percentage of CD8+T cell, CD4+T cell and theratio of CD8+T/CD4+T, but a positive correlation with Treg cell, Th17,Treg/Th17and MDSCs. correlation coefficient of0.800,0.800.0.100,-0.400,-0.800,-0.400,-0.800.Conclusions：1. MMP inhibitor improves effects of a-CTLA-4treatment for breast cancer inmouse by inhibiting tumor growth and reducing metastases.2. Compared to a-CTLA-4monotherapy, MMP inhibitor and a-CTLA-4treatment increases the percentage of CD4+T, CD8+T cell and the ratio ofCD8+T/CD4+T, reduces immunosuppressed cells, including MDSCs andTreg cell in mouse spleen obviously.3. Compared to a-CTLA-4monotherapy, MMP inhibitor and a-CTLA-4treatment inhibits the infiltration of MDSCs、Treg cell and Th17obviously,meanwhile, reduces MVD in tumor environment obviously.